Heat-shock response of the upper intertidal barnacle Balanus glandula: thermal stress and acclimation.
Intertidal organisms inhabit an interface between aquatic and terrestrial habitats where they are exposed to extreme physical conditions during low tides (Lewis, 1978; Newell, 1979). These organisms experience body temperatures that exceed the temperature of the surrounding air and regularly approach sublethal thermal limits (Helmuth, 1999; Helmuth and Hofmann, 2001; Tomanek and Sanford, 2003). Organisms residing higher in the intertidal are more likely to experience prolonged thermal and desiccation stresses than are organisms lower in the intertidal (Wolcott, 1973; Hofmann and Somero, 1995; Roberts et al., 1997; Halpin et al., 2002). One physiological adaptation to living in a stressful habitat like the intertidal is the synthesis of molecular chaperones (Feder and Hofmann, 1999; Hochachka and Somero, 2002).
Molecular chaperones such as heat-shock proteins (Hsps) act to rescue damaged proteins and prevent them from aggregating, thereby helping conserve the pool of existing proteins from irreversible damage (Parsell and Lindquist, 1993; Buchner, 1996; Fink, 1999). Though energetically expensive, the expression of molecular chaperones may protect existing protein pools during periods of acute and chronic stress and thus reduce the subsequent higher cost of de novo protein synthesis (Somero, 2002; Hartl and Hayer-Hartl, 2002; Hofmann et al., 2002).
Elevated levels of Hsps may not completely repair proteins denatured by thermal stress; some irreversible protein damage can occur. When a protein is irreversibly damaged, ubiquitin, a low-molecular-mass protein, is bound to the damaged protein, marking it for degradation by cytoplasmic proteases (Hershko and Ciechanover, 1992; Hochstrasser, 1995). In studies that measured the level of ubiquitin conjugate, the amount of irreversibly damaged protein increased as the level of stress increased in both field and laboratory studies (Lee et al., 1988; Hofmann and Somero, 1996a; Halpin et al., 2002; Spees et al., 2002).
A series of influential studies have been published on the physiological response of rocky intertidal organisms to ecologically relevant environmental stresses (Sanders et al., 1991; Sharp et al., 1994; Hofmann and Somero, 1995; Tomanek and Somero, 1999; Hamdoun et al., 2003). Most studies have focused on the responses of molluscs, and information for other abundant intertidal organisms, such as algae, urchins, or barnacles, is limited. The acorn barnacle Balanus glandula Darwin 1854, a sessile, thermotolerant, ubiquitous, and important member of the middle-to-high rocky intertidal zone along the eastern Pacific Ocean (Newell, 1979; Morris et al., 1980), is an ideal organism in which to examine the physiological response to ecologically relevant stress in a high intertidal habitat where organisms are exposed to sublethal conditions for extended times.
Molting distinguishes crustaceans from other organisms (e.g., molluscs) in the middle-to-high rocky intertidal. Molting in a crustacean involves cuticular reorganization, limb regeneration, and protein degradation (Skinner et al., 1992; Hopkins, 1993; Roer and Dillaman, 1993). Since molecular chaperones have multiple cellular functions (Lindquist, 1986; Parsell and Lindquist, 1993), the process of molting could potentially elicit an increase in Hsp levels and either confound or interact with the effects of temperature.
The heat-shock response is plastic and can be adjusted on the basis of an organism's past experience. For example, the induction temperature at which Hsp expression is turned on can be adjusted by long-term seasonal acclimatization or short-term acclimation (reviewed in Hofmann et al., 2002). However, some intertidal organisms exist at the boundary of their physiological limit to thermal stress and do not have the capacity to acclimate to higher temperatures (Stillman and Somero, 2000; Stillman, 2003; Tomanek, 2005).
We explored the heat-shock response of B. glandula after thermal stress, by addressing the following questions: (1) At what temperature does B. glandula respond physiologically to thermal stress by expressing a heat-shock response? (2) Do specimens experiencing different thermal environments in the intertidal zone differ in their responses to physiological stress? (3) Does the molt cycle affect the heat-shock response? (4) Does thermal acclimation affect the pattern of expression of heat-shock proteins in a thermally tolerant intertidal barnacle?
Materials and Methods
Unless otherwise noted, adult specimens of Balanus glandula Darwin 1854 were collected from the intertidal zone at the mouth of the South Slough Estuary in Charleston, Oregon (43[degrees]20.4'N, 124[degrees]19.4'W). The barnacles were removed from pilings under the Charleston bridge or collected from an adjacent cobble field.
Endogenous Hsp70 and ubiquitin-conjugate levels in the laboratory
In two laboratory experiments designed to examine the heat-shock response of B. glandula, we used an immunochemical assay to measure levels of endogenous Hsp70 and ubiquitin conjugate during a 16-h recovery period after thermal incubation. Field-collected barnacles were maintained in a flowing seawater table at ambient temperature for longer than one week at an average temperature (range) of 11.2 [degrees]C (11.0 to 11.3 [degrees]C) in experiment 1 and an average temperature (range) of 12.0 [degrees]C (10.8 to 13.8 [degrees]C) in experiment 2. In experiment 1, barnacles were exposed to 11, 20, and 28 [degrees]C for 5 h, which approximated low-tide emersion. In experiment 2, B. glandula was exposed to 14 [degrees]C (maximum temperature during acclimation) and 34 [degrees]C for 8.5 h. The temperature and exposure period were raised in experiment 2 in an effort to induce a heat-shock response. To mimic conditions in the intertidal zone during low tide, barnacles were not submerged in water, but the humidity was maintained at 100%. Immediately after thermal incubation, barnacles were placed in seawater at 11 [degrees]C (experiment 1) and 14 [degrees]C (experiment 2). At 0, 2, 4, 10, and 16 h after thermal incubation was terminated, the prosomas of five barnacles from each temperature treatment were dissected, frozen with liquid nitrogen, and stored at -80 [degrees]C for analyses of endogenous Hsp70 and ubiquitin conjugate (see methods outlined below).
Endogenous Hsp70 and ubiquitin-conjugate levels in the field
Two field experiments were performed to examine the endogenous levels of Hsp70 and ubiquitin conjugate in B. glandula. In the first experiment, barnacles were collected on similarly sized cobbles ([approximately equal to]10 X 10 X 5 cm, L X W X H) from the intertidal zone at 1.7 and 0.4 m above mean lower low water (MLLW); the 0.4-m site was partially shaded by a thin layer of dried algae (Ulva sp.). The mean surface temperature ([+ or -]1 SD) on three cobbles encrusted with B. glandula at the time of collection was 29.5 [degrees]C (0.4) at 1.7 m above MLLW and 23.7 [degrees]C (0.1) at 0.4 m above MLLW. In the second field experiment, similarly sized cobbles covered with barnacles were collected at 1.7 m above MLLW to measure Hsp70 levels from B. glandula on the warmer upper surface compared to the cooler underside of the cobbles. The mean surface temperature ([+ or -]1 SD) on four cobbles was 29.0 [degrees]C (0.8) on the upper surface and 24.7 [degrees]C (0.8) on the shaded underside. After collection, barnacles were placed in a flowing seawater table at an ambient temperature of 13.6 [degrees]C for 4 h. After this time, the prosoma was removed from each barnacle, immediately frozen with liquid nitrogen, and stored at -80 [degrees]C for analyses of endogenous Hsp70 and ubiquitin conjugate (see methods outlined below).
Effect of molt cycle on temperature-induced protein expression
An independent experiment was performed to examine the effect of molting on the heat-shock response. Barnacles were collected in the field during an evening low tide in December 2002 and maintained in a seawater table at 11 [degrees]C for about 12 h. After 12 h, barnacles in distinctly recognized molt stages (Davis et al., 1973) of interecdysis (stage C) or proecdysis (stage [D.sub.2-3]) were assayed, using the metabolic labeling assay outlined below, for thermally induced protein synthesis.
Acclimation and field acclimatization
To examine the effects of acclimation to temperature, B. glandula was collected on pilings in the intertidal at the mouth of the South Slough Estuary, Charleston, Oregon, and from a rocky intertidal bench on the open coast in Sunset Bay State Park, Charleston, Oregon, during August 2003; the two collection sites were separated by about 6 km. Barnacles were returned to the laboratory and acclimated in aquaria at a constant temperature of 10, 16, or 22 [degrees]C for 8 weeks. Water was changed every 4 days, and barnacles were fed the diatom Skeletonema costatum in excess. After acclimation, metabolic labeling assays were performed (see below). To test the effects of thermal stress on field-acclimatized barnacles, specimens were collected in the field during an evening low tide in August 2003, maintained in a seawater table at about 10 [degrees]C for about 12 h, and then assayed for thermally induced protein synthesis. A metabolic labeling assay was used, following the procedures outlined below.
Endogenous Hsp70 analysis (Western-blot immunochemical assay)
Endogenous Hsp70 tissue preparation and Western-blot analysis of heat-shock protein (Hsp70) followed methods and used reagents outlined by Hofmann and Somero (1995) with the following modifications. Samples were homogenized and centrifuged at 16,000 X g for 10 min at 4 [degrees]C. Sample protein concentration was determined by a Coomassie blue assay (BioRad). The supernatant was diluted 1:1 (v/v) with sodium dodecyl sulfate (SDS) sample buffer, heated for 5 min at 100 [degrees]C, and centrifuged at 16,000 X g for 30 s. An aliquot of the homogenate was stored at -80 [degrees]C for ubiquitin-conjugate analysis. From the remaining supernatant, 10 [micro]g of total protein per sample was electrophoretically separated on a 7.5% SDS-polyacrylamide gel (Laemmli, 1970). Separated proteins were transferred to a presoaked nitrocellulose membrane (0.45 [micro]m, Schleicher & Schuell) with a Mini Trans-Blot cell (BioRad), and then dried in an oven at 50 [degrees]C for 20 min. The membrane was blocked, incubated for 1.5 h with a primary antibody diluted 1:2500 (anti-Hsp70, rat monoclonal, MA3-001, Affinity Bioreagents; binds to constitutive and induced isoforms), incubated for 30 min with a secondary antibody diluted 1:2000 (rabbit anti-rat, AI-4000, Vector Laboratories), and then incubated for 1 h with protein-A horseradish peroxidase conjugate diluted 1:5000 (BioRad, 170-6522). To visualize the proteins, an enhanced chemiluminescence detection method (ECL reagents, Amersham) was used, followed by exposing the membrane to X-ray film (Kodak X-OMAT). Optical density of each visualized band was quantified using Gel-Pro Analyzer software (ver. 4.0, Media Cybernetics). When multiple gels were compared within an experiment, all bands on each gel were normalized to a positive control prepared from heat-shocked mussel (Mytilus californianus) so relative comparisons could be made.
Ubiquitin-conjugated proteins were quantified following the methods of Hofmann and Somero (1995) with the following exceptions. Samples were diluted to a concentration of 5 [micro]g [ml.sup.-1] in a saline solution, loaded in triplicate 100-[micro]l volumes onto a presoaked nitrocellulose membrane (0.45 [micro]m, Schleicher & Schuell) in a dot-blot vacuum apparatus (Bio-Rad), gravity-fed through the membrane, and dried at 50 [degrees]C for 20 min.
The membrane was blocked, incubated for 1.5 h with a polyclonal rabbit anti-ubiquitin-conjugate primary antibody diluted 1:2500 (provided by G. Hofmann, University of California, Santa Barbara), and then incubated for 1 h with protein-A horseradish-peroxidase-conjugate diluted 1:5000 (Vector Laboratories, PI-1000). Visual detection and analysis was identical to methods previously outlined for Hsp70.
Metabolic labeling assay
To quantify thermally induced protein synthesis, a series of laboratory experiments were conducted using the following general methods. Specific experimental manipulations were described in previous sections. Two right and two left depressor muscles (e.g., scutorum rostralis and scutorum lateralis) from B. glandula specimens with a basal diameter greater than 15 mm were dissected in a cold room at 10 [degrees]C. Muscle fibers were placed in a barnacle "Ringer's" solution (Hoyle and Smyth, 1963), with 10 mmol [l.sup.-1] glucose added. Each depressor muscle was assigned to one of four temperature treatments of 10, 23, 28, or 33 [degrees]C for 2.5 h. This dissection procedure allowed the in vitro exposure of muscle tissue from one individual to four temperatures. After thermal incubation, muscle tissue was incubated in 500 [micro]l of the Ringer's solution with 50 [micro]Ci of [.sup.35.S]-labeled methionine/cysteine (NEG-772, Perkin Elmer) at 10 [degrees]C for 4 h to allow adequate time for uptake of the label, washed three times in the Ringer's solution, frozen with liquid nitrogen, and stored at -20 [degrees]C for no longer than 2 weeks.
Frozen samples were homogenized in buffer consisting of 32 mmol [l.sup.-1] Tris-HCL, pH 6.8, 2% SDS, and 1 mmol [l.sup.-1] phenylmethylsulfonyl fluoride, heated to 100 [degrees]C for 5 min, and then centrifuged at 16,000 X g for 10 min. Determination of the [.sup.35.S]-methionine/cysteine incorporated into protein was by liquid scintillation counting (see Tomanek and Somero, 1999). Equivalent amounts of radiolabeled protein were separated electrophoretically on 7.5% SDS-polyacrylamide gels (Laemmli, 1970). The radioactivity of labeled protein loaded into gels varied between experiments, but never within an experiment. Typically, 60,000 counts [min.sup.-1] (cpm) were loaded, but in a few cases it was necessary to load 40,000 or 50,000 cpm. Gels were fixed in 60% distilled water, 30% methanol, and 10% acetic acid for 1 h, dried under vacuum, and then exposed to X-ray film (Kodak X-OMAT) with an intensifying screen (Kodak BioMax Transcreen LE) for 12 h (60,000 cpm), 14.5 h (50,000 cpm), or 18 h (40,000 cpm) at -80 [degrees]C. Recognizable bands in the 70-kDa and 90-kDa regions of each gel were quantified using Gel-Pro Analyzer software (Media Cybernetics). The proteins (heat-shock proteins) quantified in this series of experiments were referred to by the range of their molecular mass. Proteins in the 70-80-kDa region were referred to as Hsp70, and those in the 89-95-kDa region were referred to as Hsp90.
Data examined in this study were analyzed with Systat (ver. 9.0, SPSS Inc.) and Statistica (ver. 6.0, Statsoft). The assumptions of normality and homoscedasticity were tested with a Kolmogorov-Smirnov test with Lilliefors option and a Cochran's C test, respectively. The assumption of normality was violated a few times; however, ANOVAs are robust to departures from normality (Sokal and Rohlf, 1995; Underwood, 1997). All repeated-measures ANOVAs were subjected to Mauchly's test of sphericity (Crowder and Hand, 1990). Violations of sphericity were interpreted by using the Greenhouse-Geisser epsilon to adjust the appropriate degrees of freedom.
[FIGURE 1 OMITTED]
To test the effects of incubation temperature on endogenous Hsp70 and ubiquitin-conjugate levels (experiments 1 and 2), separate ANOVAs were performed. All dependent variables were natural-log-transformed prior to analysis.
To determine differences between endogenous Hsp70 and ubiquitin levels in field-collected barnacles, separate t-tests were performed. Data were natural-log-transformed for endogenous Hsp70 levels in both field experiments prior to analysis.
Because muscle tissue from a single barnacle was tested across all incubation temperatures in all metabolic labeling experiments, a repeated-measures ANOVA was performed with incubation temperature as the repeated measure. The analysis was performed separately for Hsp70 and Hsp90. All data were natural-log-transformed prior to analyses. In acclimation experiments, band densities from incubation temperatures of 23, 28, and 33 [degrees]C were normalized to the mean of the 10 [degrees]C control treatment so comparisons could be made between acclimations (Tomanek and Somero, 1999). Comparisons between treatments within an experiment were determined from visual observation of the data.
Endogenous Hsp70 and ubiquitin-conjugate levels in the laboratory
Only one resolvable band (75-kDa isoform) was observed for Balanus glandula in all immunochemical assays (Fig. 1). Two bands (77-kDa and 72-kDa isoforms) were resolvable in the positive control prepared from heat-shocked tissue from Mytilus californianus.
Over a 16-h period during experiment 1, Hsp70 levels did not significantly increase when B. glandula was exposed to elevated temperatures of 20 and 28 [degrees]C compared to the control temperature of 11[degrees]C (Fig. 2A, ANOVA, [F.sub.2, 72] = 0.93, P = 0.40). Levels of Hsp70 did vary significantly over time (ANOVA, [F.sub.5, 72] = 3.7, P = 0.005); however, increased or decreased levels occurred across all temperatures, including the control. A nonsignificant interaction between temperature and time further indicated that levels of Hsp70 did not vary between temperature treatments during the 16-h recovery period (ANOVA, [F.sub.10, 72] = 0.50, P = 0.892). In experiment 2, significantly higher levels of Hsp70 were observed in the 34 [degrees]C treatment compared to the 14 [degrees]C control (Fig. 3A, ANOVA, [F.sub.1, 48] = 8.10, P = 0.006). During the recovery period in experiment 2, endogenous Hsp70 levels did not change significantly (ANOVA, [F.sub.5, 48] = 0.84, P = 0.53). An interaction of temperature and time was not observed (ANOVA, [F.sub.5, 48] = 0.56, P = 0.73).
[FIGURE 2 OMITTED]
Levels of ubiquitin conjugate were measured in experiments 1 and 2 to determine whether irreversible protein damage occurred in response to thermal stress during laboratory experiments. Ubiquitin-conjugate levels did not vary significantly as a result of incubation temperature (ANOVA, [F.sub.2, 72] = 1.42, P = 0.25) or over time (ANOVA, [F.sub.5, 72] = 0.35, P = 0.88) throughout the entire 16-h recovery period after thermal incubation during experiment 1 (Fig. 2B). Ubiquitin-conjugate levels also did not vary significantly as an effect of temperature (ANOVA, [F.sub.1, 48] = 0.84, P = 0.36) or over the course of recovery (ANOVA, [F.sub.5, 48] = 0.80, P = 0.55) after exposure to 34 [degrees]C for 8.5 h during experiment 2 (Fig. 3B).
Endogenous Hsp70 and ubiquitin-conjugate levels in the field
Endogenous levels of Hsp70 from specimens of B. glandula collected on cobbles from 1.7 m and 0.4 m above mean lower low water (MLLW) were not significantly different (t = 1.54, P = 0.17). In addition, there was no significant difference between ubiquitin-conjugate levels in B. glandula collected from the high intertidal and mid-intertidal zones (t = -0.42, P = 0.68). In the second field experiment, endogenous Hsp70 levels in B. glandula attached to the warmer top and cooler underside of the cobbles were not significantly different (t = -1.40, P = 0.22).
Effect of molt cycle on temperature-induced protein expression
Comparisons of Hsp70 and Hsp90 levels between two prominent molt-cycle stages (i.e., interecdysis and proecdysis) suggest that molt stage did not affect the levels of induced heat-shock protein expression across incubation temperatures (Fig. 4, Table 1). A significant increase in Hsp70 levels was observed as incubation temperature increased (Fig. 4A, Table 1). Levels of Hsp90 also significantly varied over incubation temperature (Table 1). However, unlike the Hsp70 pattern in which levels continued to increase as incubation temperature increased, Hsp90 levels reached a maximum at 28 [degrees]C and then decreased at 33 [degrees]C (Fig. 4B). The autoradiograph image in Figure 5 represents a typical response of B. glandula muscle tissue to thermal stress in this and other metabolic labeling assays.
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
Acclimation and field acclimatization
The effects of acclimation and acclimatization on the heat-shock response differed between the two populations of B. glandula examined during August 2003. For the Charleston (CH) population of B. glandula, Hsp70 levels varied significantly as a result of acclimation temperature (Table 2). Barnacles acclimated to 16 [degrees]C and 22 [degrees]C displayed higher levels of Hsp70 across all incubation temperatures compared to barnacles that were field-acclimatized or acclimated to 10 [degrees]C (Fig. 6A). In addition, the field-acclimatized barnacles from the CH population displayed patterns of Hsp70 synthesis that were similar to those in the 10 [degrees]C acclimation treatment (Fig. 6A).
Although levels of Hsp70 in the Sunset Bay (SB) population did significantly increase as incubation temperature increased, those levels were not affected by acclimation temperature alone (Fig. 6B, Table 2). Acclimation temperature interacted significantly with incubation temperature such that levels of Hsp70 for barnacles acclimated to 10 [degrees]C did not increase as incubation temperature increased, whereas Hsp70 levels in all other acclimatization and acclimation treatments did increase with incubation temperature (Fig. 6B, Table 2). At an incubation temperature of 28 [degrees]C, field-acclimatized barnacles from the SB population displayed a pattern similar to those in the 10 and 16 [degrees]C acclimation treatment; but at an incubation temperature of 33 [degrees]C, the field-acclimatized Hsp70 level was similar to that in the 22 [degrees]C acclimation treatment (Fig. 6B).
Levels of Hsp90 did not vary significantly as a function of acclimation or incubation temperature in the CH population; no recognizable patterns were observed (Fig. 6C, Table 2). In contrast, the effect of acclimation on Hsp90 levels did vary significantly in the SB population; Hsp90 levels tended to decrease as acclimation temperature increased (Fig. 6D, Table 2). However, Hsp90 levels in the SB population did not vary significantly across incubation temperatures (Table 2).
Intertidal organisms in the Pacific Northwest regularly experience body temperatures from 20 to 35 [degrees]C during low tides (Tomanek and Somero, 1999; Hamdoun et al., 2003; Fitzhenry et al., 2004). Metabolic labeling experiments in this study (Figs. 4 and 6) indicated that Hsp70 is induced in Balanus glandula at 23 [degrees]C, regardless of acclimatization or acclimation, indicating that heat-shock proteins are upregulated in this species on a regular basis.
[FIGURE 5 OMITTED]
In B. glandula, the pattern of Hsp90 synthesis differed from that of Hsp70. For specimens examined in December 2002, maximum expression of Hsp90 was between 23 and 28 [degrees]C and then decreased at 33 [degrees]C; in comparison, Hsp70 remained at maximum expression levels above 28 [degrees]C (Fig. 4). Presumably, at temperatures above 33 [degrees]C the expression of Hsp90 approached a negligible level. A difference in the patterns of expression of the two heat-shock proteins is not surprising, as different molecular chaperones perform different functions within a cell (Lindquist, 1986; Parsell and Lindquist, 1993).
Levels of molecular chaperones have been shown to increase in some intertidal molluscs during the recovery period after thermal stress (Hofmann and Somero, 1996b; Tomanek and Somero, 2000). A reduction in metabolic rate during aerial exposure and a resultant decrease in protein synthesis have been suggested as the mechanism regulating the lag time in Hsp synthesis in bivalve molluscs (see Hofmann and Somero, 1996b). No lag time for maximal expression of Hsp70 was seen in B. glandula in the 16 h after thermal stress. Our results suggest that levels of endogenous Hsp70 were either high before thermal stress or were synthesized during the stress period.
Immunochemical assays are very effective for measuring heat-shock response in a variety of molluscs (Hofmann and Somero, 1995; Dahlhoff et al., 2001; Tomanek and Sanford, 2003; Sorte and Hofmann, 2005), but they may be a less effective measurement method in the crustacean B. glandula. In Mytilus edulis, immunochemical assays that used the same antibody as in the present study identified two obvious Hsp70 isoforms, presumably constitutive and induced isoforms (Hofmann and Somero, 1995; Halpin et al., 2002). Typically, a cell will synthesize both constitutive and inducible isoforms of molecular chaperones (Sanders, 1993; Feder and Hofmann, 1999). Only one resolvable band, possibly consisting of both constitutive and inducible Hsp70, was visible in B. glandula. Therefore, relatively high constitutive levels may have made the resolution of induced levels within the same protein band difficult to observe. This would explain why a heat-shock response was observed with the immunochemical method only after prolonged exposure to 34 [degrees]C (presumably an extreme level of stress). An alternative is that the antibody we used was not specific to the inducible Hsp70 isoform. However, this is an unlikely explanation since an induced response was observed during experiment 2 (Fig. 3). If an immunochemical assay is to be employed for future work on B. glandula, two-dimensional gel electrophoresis should be used to separate the constitutive from the induced isoform.
[FIGURE 6 OMITTED]
Two desert ant species that regularly experience temperatures above 50 [degrees]C while foraging express heat-shock proteins at a temperature as low as 25 [degrees]C (Gehring and Wehner, 1995). Gehring and Wehner (1995) suggest that the ants synthesize Hsps at relatively low temperatures prior to exposure to high temperatures as an adaptation to foraging in a high-temperature habitat. Immunochemical assays on B. glandula indicated high levels of endogenous Hsp70 at control temperatures. A correlation between thermotolerance and constitutive Hsp70 levels has been observed in marine snails (Sorte and Hofmann, 2004). Therefore, high levels of endogenous Hsp70 might suggest that B. glandula is preparing for the physiological stress it will potentially encounter during low tide.
Endogenous levels of Hsp70 were not significantly different for B. glandula individuals collected from intertidal elevations of 1.7 m and 0.4 m above mean lower low water (MLLW) or for those from the tops and undersides of cobbles at 1.7 m above MLLW. A higher intertidal height is usually more stressful because the intensity of the thermal stress is in part a function of the change in temperature multiplied by the duration of exposure (Hochachka and Somero, 2002). We expected higher levels of endogenous Hsp70 expression in habitats presumed to be more stressful (1.7 m vs. 0.4 m and top vs. bottom of cobbles). For Mytilus californianus, Roberts et al. (1997) reported higher levels of endogenous Hsp70 in individuals collected higher in the intertidal zone than in those collected lower in the zone. Similarly, for M. trossulus, Hsp70 levels were higher for individuals from a more stressful intertidal habitat compared to a subtidal habitat (Hofmann and Somero, 1995). In both field experiments with barnacles, recorded temperatures were high enough to elicit a heat-shock response in the more stressful habitats of the high intertidal zone (1.7 m above MLLW) or cobble topside. Constitutively expressed Hsp70 isoforms may have masked any heat-induced isoforms in barnacles from the high intertidal and on the top of the cobble.
When B. glandula experienced temperatures as high as 34 [degrees]C for 8.5 h in the laboratory or when specimens encountered high temperatures in the field, they did not show evidence of irreversible protein damage. These results differ from other studies that have examined levels of ubiquitin conjugate after thermal stress. In multiple cases, an increase in ubiquitin conjugate levels and subsequent irreversible protein damage occurred for Drosophila (Lee et al., 1988), intertidal mussels (Hofmann and Somero, 1995, 1996a; Buckley et al., 2001; Halpin et al., 2002), and lobster (Spees et al., 2002) when they experienced physiologically stressful conditions and showed a heat-shock response. Additionally, levels of ubiquitin conjugate were elevated when Balanus crenatus, a subtidal congener of B. glandula, was exposed to a temperature of 26 [degrees]C (M. Berger, unpubl. data). These results from organisms other than B. glandula suggest that although heat-shock proteins were expressed at elevated levels, some irreversible protein damage occurred. A heat-shock response occurred in B. glandula above 23 [degrees]C, yet there was no indication of irreversible protein damage. This result implies that B. glandula is well adapted to living in a stressful habitat and may have a concentration of heat-shock proteins adequate to remediate protein denaturation. Further work correlating the relationship between Hsp expression and irreversible protein damage in both laboratory and field-collected specimens is necessary.
We found that the expression of Hsp70 and Hsp90 varied as a function of temperature, but not as a function of the two molt stages examined. This result differs from other work showing an increase in levels of Hsp90 mRNA in lobster (Homarus americanus) claw muscle tissue during stage [D.sub.2] of molting (Spees et al., 2003) and in lobster midgut tissue injected with a molting hormone (Chang et al., 1999). Our finding that molt stage did not affect levels of heat-shock protein in B. glandula suggests that molting barnacles will not be more susceptible than non-molting barnacles to deleterious physiological effects of thermal stress.
Increases in acclimation or acclimatization temperature are typically associated with upward shifts in the induction temperature of heat-shock proteins (Dietz and Somero, 1992; Tomanek and Somero, 1999; Buckley et al., 2001; Buckley and Hofmann, 2002). This was not the case for acclimated or summer-acclimatized specimens of B. glandula; an upward shift in the Hsp induction temperature did not occur. In fact, barnacles acclimated to higher temperatures (see Fig. 6) showed higher levels of Hsp70, which was contrary to the expectation that an organism acclimated to lower temperatures will be more sensitive to increased thermal stress. Recent studies suggest that some thermotolerant intertidal organisms may be the least plastic in their ability to acclimate to higher temperatures (Stillman, 2003; Stenseng et al., 2005; Tomanek, 2005). Because B. glandula regularly experiences high temperatures for an extended time, it may be operating at a maximal level without the ability to further modify a physiological response. If the predication is correct, thermally tolerant species like B. glandula may be more sensitive to increased global temperatures than previously expected, especially at the southern limit of their geographic distributions.
We thank G. Hofmann and M. Ryan for technical help in the laboratory. We also thank A. Helms for help in maintaining organisms in the laboratory. Earlier drafts of this manuscript were improved from comments by L. Burnett, I. McGaw, P. Phillips, A. Shanks, N. Terwilliger, and two anonymous reviewers. This research was supported by NSF grant OCE-9911682 to R. Emlet, NSF-IGERT grant 9972830 to J. Postlethwait, and ERD-OCRM-NOAA grant NA17OR1172 to M. Berger. This research was performed in partial fulfillment of a Ph.D. in the Department of Biology, University of Oregon.
Buchner, J. 1996. Supervising the fold: functional principles of molecular chaperones. FASEB J. 10: 10-19.
Buckley, B. A., and G. E. Hofmann. 2002. Thermal acclimation changes DNA-binding activity of heat shock factor 1 (HSF1) in the goby Gillichthys mirabilis: implications for plasticity in the heat-shock response in natural populations. J. Exp. Biol. 205: 3231-3240.
Buckley, B. A., M. Owen, and G. E. Hofmann. 2001. Adjusting the thermostat: the threshold induction temperature for the heat-shock response in intertidal mussels (genus Mytilus) changes as a function of thermal history. J. Exp. Biol. 204: 3571-3579.
Chang, E. S., S. A. Chang, R. Keller, P. S. Reddy, M. J. Snyder, and J. L. Spees. 1999. Quantification of stress in lobsters: crustacean hyperglycemic hormone, stress proteins and gene expression. Am. Zool. 39: 487-495.
Crowder, M. J., and D. J. Hand. 1990. Analysis of Repeated Measures. Chapman and Hall, London.
Dahlhoff, E. P., B. A. Buckley, and B. A. Menge. 2001. Physiology of the rocky intertidal predator Nucella ostrina along an environmental stress gradient. Ecology 82: 2816-2829.
Davis, C. W., U. E. H. Fyhn, and H. J. Fyhn. 1973. The intermolt cycle of cirripeds: criteria for its stages and its duration in Balanus amphitrite. Biol. Bull. 145: 310-322.
Dietz, T. J., and G. N. Somero. 1992. The threshold induction temperature of the 90-kDa heat shock protein is subject to acclimatization in eurythermal goby fishes (genus Gillichthys). Proc. Natl. Acad. Sci. USA 89: 3389-3393.
Feder, M. E., and G. E. Hofmann. 1999. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61: 243-282.
Fink, A. L. 1999. Chaperone-mediated protein folding. Physiol. Rev. 79: 425-449.
Fitzhenry, T., P. M. Halpin, and B. Helmuth. 2004. Testing the effects of wave exposure, site, and behavior on intertidal mussel body temperatures: applications and limits of temperature logger design. Mar. Biol. 145: 339-349.
Gehring, W. J., and R. Wehner. 1995. Heat shock protein synthesis and thermotolerance in Cataglyphis, an ant from the Sahara desert. Proc. Natl. Acad. Sci. USA 92: 2994-2998.
Halpin, P. M., C. J. Sorte, G. E. Hofmann, and B. A. Menge. 2002. Patterns of variation in levels of Hsp70 in natural rocky shore populations from microscales to mesoscales. Integr. Comp. Biol. 42: 815-824.
Hamdoun, A. M., D. P. Cheney, and G. N. Cherr. 2003. Phenotypic plasticity of HSP70 and HSP70 gene expression in the Pacific oyster (Crassostrea gigas): implications for the thermal limits and induction of thermal tolerance. Biol. Bull. 205: 160-169.
Hartl, F. U., and M. Hayer-Hartl. 2002. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295: 1852-1858.
Helmuth, B. 1999. Thermal biology of rocky intertidal mussels: quantifying body temperatures using climatological data. Ecology 80: 15-34.
Helmuth, B. S. T., and G. E. Hofmann. 2001. Microhabitats, thermal heterogeneity, and patterns of physiological stress in the rocky intertidal zone. Biol. Bull. 201: 374-384.
Hershko, A., and A. Ciechanover. 1992. The ubiquitin system for protein degradation. Annu. Rev. Biochem. 61: 761-807.
Hochachka, P. W., and G. N. Somero. 2002. Biochemical Adaptation: Mechanism and Process in Physiological Evolution. Oxford University Press, New York.
Hochstrasser, M. 1995. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr. Opin. Cell Biol. 7: 215-223.
Hofmann, G. E., and G. N. Somero. 1995. Evidence for protein damage at environmental temperatures: seasonal changes in levels of ubiquitin conjugates and hsp70 in the intertidal mussel Mytilus trossulus. J. Exp. Biol. 198: 1509-1518.
Hofmann, G. E., and G. N. Somero. 1996a. Interspecific variation in thermal denaturation of proteins in the congeneric mussels Mytilus trossulus and M. galloprovincialis: evidence from the heat-shock response and protein ubiquitination. Mar. Biol. 126: 65-75.
Hofmann, G. E., and G. N. Somero. 1996b. Protein ubiquitination and stress protein synthesis in Mytilus trossulus occurs during recovery from tidal emersion. Mol Mar. Biol. Biotechnol. 5: 175-184.
Hofmann, G. E., B. A. Buckley, S. P. Place, and M. L. Zippay. 2002. Molecular chaperones in ectothermic marine animals: biochemical function and gene expression. Integr. Comp. Biol. 42: 808-814.
Hopkins, P. M. 1993. Regeneration of walking legs in the fiddler crab Uca pugilator. Am. Zool. 33: 348-356.
Hoyle, G., and T. J. Smyth. 1963. Neuromuscular physiology of giant muscle fibers of a barnacle, Balanus nubilus Darwin. Comp. Biochem. Physiol. 10: 291-314.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
Lee, H., J. A. Simon, and J. T. Lis. 1988. Structure and expression of ubiquitin genes of Drosophila melanogaster. Mol. Cell. Biol. 8: 4727-4735.
Lewis, J. R. 1978. The Ecology of Rocky Shores. Hodder and Stoughton, London.
Lindquist, S. 1986. The heat-shock response. Annu. Rev. Biochem. 55: 1151-1191.
Morris, R. H., D. P. Abbott, and E. C. Haderlie. 1980. Intertidal Invertebrates of California. Stanford University Press, Stanford, CA.
Newell, R. C. 1979. Biology of Intertidal Animals. Marine Ecological Surveys, Faversham, United Kingdom.
Parsell, D., and S. Lindquist. 1993. The function of heat-shock proteins in stress tolerance degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27: 437-796.
Roberts, D. A., G. E. Hofmann, and G. N. Somero. 1997. Heat-shock protein expression in Mytilus californianus: acclimatization (seasonal and tidal-height comparisons) and acclimation effects. Biol. Bull. 192: 309-320.
Roer, R. D., and R. M. Dillaman. 1993. Molt-related change in integumental structure and function. Pp. 1-37 in The Crustacean Integument: Morphology and Biochemistry, M. N. Horst and J. A. Freeman, eds. CRC Press, Boca Raton, FL.
Sanders, B. M. 1993. Stress proteins in aquatic organisms: an environmental perspective. Crit. Rev. Toxicol. 23: 49-75.
Sanders, B. M., C. Hope, V. M. Pascoe, and L. S. Martin. 1991. Characterization of the stress protein response in two species of Collisella limpets with different temperature tolerances. Physiol. Zool. 64: 1471-1489.
Sharp, V. A., D. Miller, J. C. Bythell, and B. E. Brown. 1994. Expression of low molecular weight HSP70 related polypeptides from a symbiotic sea anemone Anemonia viridis Forskall in response to heat shock. J. Exp. Mar. Biol. Ecol. 179: 179-193.
Skinner, D. M., S. S. Kumari, and J. J. O'Brien. 1992. Proteins of the crustacean exoskeleton. Am. Zool. 32: 470-484.
Sokal, R. R., and J. Rohlf. 1995. Biometry. W.H. Freeman, New York.
Somero, G. N. 2002. Thermal physiology and vertical zonation of intertidal animals: optima, limits, and costs of living. Integr. Comp. Biol. 42: 780-789.
Sorte, C. J. B., and G. E. Hofmann. 2004. Changes in latitudes, changes in aptitudes: Nucella canaliculata (Mollusca: Gastropoda) is more stressed at its range edge. Mar. Ecol. Prog. Ser. 274: 263-268.
Sorte, C. J. B., and G. E. Hofmann. 2005. Thermotolerance and heat-shock protein expression in Northeastern Pacific Nucella species with different biogeographical ranges. Mar. Biol. 146: 985-993.
Spees, J. L., S. A. Chang, M. J. Snyder, and E. S. Chang. 2002. Thermal acclimation and stress in the American lobster, Homarus americanus: equivalent temperature shifts elicit unique gene expression patterns for molecular chaperones and polyubiquitin. Cell Stress Chaperones 7: 97-106.
Spees, J. L., S. A. Chang, D. L. Mykles, M. J. Snyder, and E. S. Chang. 2003. Molt cycle-dependent molecular chaperone and polyubiquitin gene expression in lobster. Cell Stress Chaperones 8: 258-264.
Stenseng, E., C. E. Braby, and G. N. Somero. 2005. Evolutionary and acclimation-induced variation in the thermal limits of heart function in congeneric marine snails (genus Tegula): implications for vertical zonation. Biol. Bull. 208: 138-144.
Stillman, J. H. 2003. Acclimation capacity underlies susceptibility to climate change. Science 301: 65.
Stillman, J. H., and G. N. Somero. 2000. A comparative analysis of the upper thermal tolerance limits of eastern Pacific Porcelain crabs, genus Petrolisthes: influences of latitude, vertical zonation, acclimation, and phylogeny. Physiol. Biochem. Zool. 73: 200-208.
Tomanek, L. 2005. Two-dimensional gel analysis of the heat-shock response in marine snails (genus Tegula): interspecific variation in protein expression and acclimation ability. J. Exp. Biol. 208: 3133-3143.
Tomanek, L., and E. Sanford. 2003. Heat-shock protein70 (Hsp70) as a biochemical stress indicator: an experimental field test in two congeneric intertidal gastropods (genus: Tegula). Biol. Bull. 205: 276-284.
Tomanek, L., and G. N. Somero. 1999. Evolutionary and acclimation-induced variation in the heat-shock responses of congeneric marine snails (genus Tegula) from different thermal habitats: implications for limits of thermotolerance and biogeography. J. Exp. Biol. 202: 2925-2936.
Tomanek, L., and G. N. Somero. 2000. Time course and magnitude of synthesis of heat-shock proteins in congeneric marine snails (genus Tegula) from different tidal heights. Physiol. Biochem. Zool. 73: 249-256.
Underwood, A. J. 1997. Experiments in Ecology: Their Logical Design and Interpretation Using Analysis of Variance. Cambridge University Press, Cambridge.
Wolcott, T. G. 1973. Physiological ecology and intertidal zonation in limpets (Acmaea): a critical look at "limiting factors." Biol. Bull. 145: 389-422.
MICHAEL S. BERGER (*,1) AND RICHARD B. EMLET
Oregon Institute of Marine Biology, University of Oregon, Charleston, Oregon 97420
Received 14 March 2006; accepted 20 February 2007.
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
(1) Current address: Ecology and Evolution, University of California, Irvine, 321 Steinhaus Hall, Irvine, CA 92697.
Table 1 Repeated-measures ANOVA on the effect of molt stage on temperature-induced Hsp70 and Hsp90 levels in Balanus glandula (see Fig. 4) Sum of Mean- Source* squares df square F-ratio P Hsp70 Between subjects Molt stage 0.816 1 0.816 0.984 0.350 Error 8.300 10 0.830 Within subjects (1) Temperature 61.132 3 20.377 20.337 0.000 Temperature X molt-stage 0.735 3 0.245 0.245 0.740 Error 30.060 30 1.002 Hsp90 Between subjects Molt stage 0.003 1 0.003 0.001 0.970 Error 28.079 10 2.808 Within subjects (2) Temperature 46.058 3 15.353 10.783 0.001 Temperature X molt-stage 7.024 3 2.341 1.645 0.220 Error 42.712 30 1.424 * The assumption of sphericity (Mauchly's test) was violated in all repeated-measures ANOVAs. The degrees of freedom were multiplied by the Greenhouse-Geisser epsilon, and an adjusted P value was calculated. The adjusted P values are reported. Greenhouse-Geisser epsilon values for within subjects: (1) 0.5275 and (2) 0.6006. Table 2 Repeated-measures ANOVA on the effect of acclimation on temperature-induced Hsp70 and Hsp90 levels from Balanus glandula collected during the summer from Charleston population and Sunset Bay population (see Fig. 6) Sum of Mean- Source squares df square F-ratio P Hsp70 -- Charleston population Between subjects Acclimation 24.742 3 8.247 3.704 0.023 Error 62.344 28 2.227 Within subjects Temperature 27.806 2 13.903 33.763 0.000 Temperature X acclimation 1.001 6 0.168 0.408 0.870 Error 23.060 56 0.412 Hsp70 -- Sunset Bay population Between subjects Acclimation 12.236 3 4.079 1.230 0.320 Error 98.811 28 3.315 Within subjects Temperature 39.011 2 19.506 28.266 0.000 Temperature X acclimation 9.630 6 1.605 2.326 0.045 Error 38.645 56 0.690 Hsp90 -- Charleston population Between subjects Acclimation 30.069 3 10.023 0.480 0.700 Error 584.965 28 20.891 Within subjects Temperature 2.720 2 1.364 0.113 0.890 Temperature X acclimation 134.319 6 22.387 1.847 0.110 Error 678.624 56 12.118 Hsp90 -- Sunset Bay population Between subjects Acclimation 9.218 3 3.073 3.291 0.035 Error 26.142 28 0.934 Within subjects Temperature 0.123 2 0.061 0.551 0.580 Temperature X acclimation 0.360 6 0.060 0.539 0.780 Error 6.243 56 0.111
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|Author:||Berger, Michael S.; Emlet, Richard B.|
|Publication:||The Biological Bulletin|
|Date:||Jun 1, 2007|
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